Discovery of Antibacterial Piperazic Acid-containing Pseudopeptides from a Saccharothrix Strain Using Targeted Genetic Screening and Molecular Networking Strategies

Piperazic acid (Piz)-containing compounds are a distinctive class of microbial metabolites characterized by a unique N–N bond and diverse bioactivities, rendering them as promising scaffolds for drug discovery. Herein, we utilized an integrated discovery strategy combining PCR-based genetic screening, genome mining and MS/MS-based molecular networking to target Piz-containing metabolites from an actinomycetes library. This effort led to the isolation of nine new Piz-containing linear pseudopeptides, saccharothriotides A–I (1–9), along with the known compound Sch 382583 (10), from a desert-derived actinomycete Saccharothrix sp. 275. Their planar structures and absolute configurations were elucidated by spectroscopic analysis, advanced Marfey’s method, phenylglycine methyl ester (PGME) derivatization, and biosynthetic pathway deduction. Bioactivity assays revealed compounds 1–3, which feature a hydroxamic acid moiety, exhibited significant activity against Gram-positive pathogens (MIC = 0.5–16 μg/mL). Notably, antibacterial efficacy of 1–3 against vancomycin-resistant Enterococcus faecium (MIC = 2–4 μg/mL) markedly outperformed the antibiotic levofloxacin (MIC = 64 μg/mL). They also moderately inhibited Gram-negative bacteria such as Escherichia coli and Acinetobacter baumannii (MIC = 16–64 μg/mL). As the first Piz-containing metabolites reported from the genus Saccharothrix, this work underscores the effectiveness of combining genetic and metabolomic strategies for discovering bioactive natural products from underexplored microorganisms.